Bottom Line:
A high level of genetic diversity was found within the 13 isolates.This suggests that the vegetative incompatibility loci within B. cinerea are associated with regions of increased genetic diversity.This suggests that B. cinerea does not display an elevated spontaneous mutation rate.

ABSTRACTHow standing genetic variation within a pathogen contributes to diversity in host/pathogen interactions is poorly understood, partly because most studied pathogens are host-specific, clonally reproducing organisms which complicates genetic analysis. In contrast, Botrytis cinerea is a sexually reproducing, true haploid ascomycete that can infect a wide range of diverse plant hosts. While previous work had shown significant genomic variation between two isolates, we proceeded to assess the level and frequency of standing variation in a population of B. cinerea. To begin measuring standing genetic variation in B. cinerea, we re-sequenced the genomes of 13 different isolates and aligned them to the previously sequenced T4 reference genome. In addition one of these isolates was resequenced from four independently repeated cultures. A high level of genetic diversity was found within the 13 isolates. Within this variation, we could identify clusters of genes with major effect polymorphisms, i.e., polymorphisms that lead to a predicted functional knockout, that surrounded genes involved in controlling vegetative incompatibility. The genotype at these loci was able to partially predict the interaction of these isolates in vegetative fusion assays showing that these loci control vegetative incompatibility. This suggests that the vegetative incompatibility loci within B. cinerea are associated with regions of increased genetic diversity. The genome re-sequencing of four clones from the one isolate (Grape) that had been independently propagated over 10 years showed no detectable spontaneous mutation. This suggests that B. cinerea does not display an elevated spontaneous mutation rate. Future work will allow us to test if, and how, this diversity may be contributing to the pathogen's broad host range.

Figure 1: Allele diversity in comparison to reference T4. The number of isolates that share a polymorphic allele in comparison to the reference T4 isolate was plotted against the total number of polymorphisms with that level of diversity.

Mentions:
To identify genome-wide polymorphism levels in the nuclear genome, we aligned the genomes of the above isolates to the previously sequenced reference T4. This analysis identified 980,329 unique SNPs and 157,936 unique Indels across all isolates with between 144,109 and 403,608 polymorphisms per isolate (Table 1). There was no significant correlation between the level of sequence variation in an isolate and the depth of sequencing (Table 1, P = 0.09, R = 0.52, N = 12). Any appearance of a correlation was solely driven by the Grape isolate being both more sequenced and generally more diverged. Excluding this single outlier abolished any relationship between sequence variation and sequencing depth (Table 1, P = 0.99, R = 0.002, N =11). Aligning to the T4 reference also found extensive insertion and deletion polymorphisms. On average there were 12,560 deletions (7838–21,166) and 13,966 insertions (8492–25,137) per isolate in comparison to T4 (Table 1). Combining the polymorphisms (both SNP and indel) from all pairwise combinations of isolates yielded an average of at least one polymorphism between the isolates every 36 bp, giving an average density of 28 polymorphisms per kb within the population. Plotting the number of isolates that share a specific polymorphism in comparison to T4 showed that most alleles were of minor frequency with the major peak being a single isolate having each specific difference in comparison to T4 (Figure 1). These estimates for genomic diversity within B. cinerea are higher than that found so far in species with detailed whole genome resequencing projects such as Arabidopsis (Cao et al., 2011; Long et al., 2013), Humans (Altshuler et al., 2010), and Yeast (Schacherer et al., 2009).

Figure 1: Allele diversity in comparison to reference T4. The number of isolates that share a polymorphic allele in comparison to the reference T4 isolate was plotted against the total number of polymorphisms with that level of diversity.

Mentions:
To identify genome-wide polymorphism levels in the nuclear genome, we aligned the genomes of the above isolates to the previously sequenced reference T4. This analysis identified 980,329 unique SNPs and 157,936 unique Indels across all isolates with between 144,109 and 403,608 polymorphisms per isolate (Table 1). There was no significant correlation between the level of sequence variation in an isolate and the depth of sequencing (Table 1, P = 0.09, R = 0.52, N = 12). Any appearance of a correlation was solely driven by the Grape isolate being both more sequenced and generally more diverged. Excluding this single outlier abolished any relationship between sequence variation and sequencing depth (Table 1, P = 0.99, R = 0.002, N =11). Aligning to the T4 reference also found extensive insertion and deletion polymorphisms. On average there were 12,560 deletions (7838–21,166) and 13,966 insertions (8492–25,137) per isolate in comparison to T4 (Table 1). Combining the polymorphisms (both SNP and indel) from all pairwise combinations of isolates yielded an average of at least one polymorphism between the isolates every 36 bp, giving an average density of 28 polymorphisms per kb within the population. Plotting the number of isolates that share a specific polymorphism in comparison to T4 showed that most alleles were of minor frequency with the major peak being a single isolate having each specific difference in comparison to T4 (Figure 1). These estimates for genomic diversity within B. cinerea are higher than that found so far in species with detailed whole genome resequencing projects such as Arabidopsis (Cao et al., 2011; Long et al., 2013), Humans (Altshuler et al., 2010), and Yeast (Schacherer et al., 2009).

Bottom Line:
A high level of genetic diversity was found within the 13 isolates.This suggests that the vegetative incompatibility loci within B. cinerea are associated with regions of increased genetic diversity.This suggests that B. cinerea does not display an elevated spontaneous mutation rate.

ABSTRACTHow standing genetic variation within a pathogen contributes to diversity in host/pathogen interactions is poorly understood, partly because most studied pathogens are host-specific, clonally reproducing organisms which complicates genetic analysis. In contrast, Botrytis cinerea is a sexually reproducing, true haploid ascomycete that can infect a wide range of diverse plant hosts. While previous work had shown significant genomic variation between two isolates, we proceeded to assess the level and frequency of standing variation in a population of B. cinerea. To begin measuring standing genetic variation in B. cinerea, we re-sequenced the genomes of 13 different isolates and aligned them to the previously sequenced T4 reference genome. In addition one of these isolates was resequenced from four independently repeated cultures. A high level of genetic diversity was found within the 13 isolates. Within this variation, we could identify clusters of genes with major effect polymorphisms, i.e., polymorphisms that lead to a predicted functional knockout, that surrounded genes involved in controlling vegetative incompatibility. The genotype at these loci was able to partially predict the interaction of these isolates in vegetative fusion assays showing that these loci control vegetative incompatibility. This suggests that the vegetative incompatibility loci within B. cinerea are associated with regions of increased genetic diversity. The genome re-sequencing of four clones from the one isolate (Grape) that had been independently propagated over 10 years showed no detectable spontaneous mutation. This suggests that B. cinerea does not display an elevated spontaneous mutation rate. Future work will allow us to test if, and how, this diversity may be contributing to the pathogen's broad host range.